Human testis specific serine/threonine kinase 1 and 2

ABSTRACT

The present invention relates to a family of testis specific kinases (the tssk family), nucleic acid sequences encoding those kinases and antibodies against the kinases. The invention is further directed to the use of those kinases as targets for isolating specific inhibitors or antagonists of tssk kinase activity. Such inhibitors are anticipated to have use as contraceptive agents.

CLAIM TO PRIORITY

This application is a continuation of International Patent ApplicationNo. PCT/US01/46803, filed on Nov. 9, 2001 and published under PCTArticle 21(2) in English, and claims priority under 35 USC §119(e) toU.S. Provisional Application Ser. Nos. 60/246,939, filed Nov. 9, 2000and 60/264,921, filed Jan. 30, 2001, the disclosures of which areincorporated herein.

US GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant Nos. HD 06274, HD 22732, HD 38082, CA 06927, awarded by NationalInstitutes of Health. The United States Government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention is directed to a family of sperm specific kinases(tssk) genes, their respective encoded proteins and antibodies againstthose proteins. The present invention also encompasses the use of thetssk kinases as targets for identifying inhibitors of tssk kinaseactivity.

BACKGROUND OF THE INVENTION

Spermatogenesis, the process in which functional sperm cells areproduced in the testis, involves specific interaction between thedeveloping germ cells and their supporting Sertoli cells as well ashormonal regulation by the androgen-producing Leydig cells. The generalorganization of spermatogenesis is essentially the same in all mammalsand can be divided into three distinct phases: 1) The initial phase isthe proliferative or spermatogonial phase during which spermatogoniaundergo mitotic division and generate a pool of spermatocytes; 2) themeiotic phase, that yields the haploid spermatids; and 3) spermiogenesiswhereby each round spermatid differentiates into a spermatozoon.Although the molecular mechanisms regulating the first two phases havebeen relatively well characterized, the molecular basis ofspermiogenesis is largely unknown.

Mammalian spermiogenesis, the postmeiotic phase of spermatogenesis, ischaracterized by dramatic morphological changes that occur in thehaploid spermatid. Some of these changes include the formation of theacrosome and its contents, the condensation and reorganization of thechromatin, the elongation and species-specific reshaping of the cell,and the assembly of the flagellum. These events result from changes inboth gene transcription and protein translation that occurs during thisdevelopmental period. Some of the proteins translated in the haploidspermatid will remain in the morphologically mature sperm after itleaves the testis. Taking this into consideration, proteins that aresynthesized during spermatogenesis might be necessary for spermatiddifferentiation and/or for sperm function during fertilization.

The present invention relates to signaling events in mammalian spermthat regulate the functions of this highly differentiated cell. Moreparticularly the invention relates to signal transduction that modulatesthe acquisition of sperm fertilizing capacity. After ejaculation, spermare able to move actively but lack fertilizing competence. They acquirethe ability to fertilize in the female genital tract in a time-dependentprocess called capacitation. Capacitation has been demonstrated to beaccompanied by the protein phosphorylation of several proteins on bothserine/threonine and tyrosine residues, and that protein tyrosinephosphorylation is regulated downstream by a cAMP/PKA pathway thatinvolves the crosstalk between these two signaling pathways. With theexception of PKA, the other kinase(s) involved in the regulation ofcapacitation are still unknown.

Additional protein kinases have been shown to be involved inspermatogenesis, however, only a few of them are exclusively expressedin germ cells or in the testis (Jinno et al, 1993, Cell Biol 13,4146-56; Nayak et al, 1998, Mech Dev 74, 171-4; Shalom & Don, 1999, MolReprod Dev 52, 392-405; Toshima et al, 1998, Biochem Biophys Res Commun249, 107-12; Toshima et al, 1999, J Biol Chem 274, 12171-6; Tseng et al,1998, DNA Cell Biol 17, 823-33; Walden & Cowan, 1993, Mol Cell Biol 13,7625-35). Examples of testis-specific kinases are the recently describedmouse genes, tssk 1, 2 and 3 (Bielke et al., 1994, Gene 139, 235-9;Kueng et al, 1997, J Cell Biol 139, 1851-9; Zuercher et al., 2000, MechDev 93, 175-7). The function of the tssk kinase family is unknown.However, since the members of this family are expressed postmeioticallyduring spermiogenesis, it is hypothesized that they have a role in germcell differentiation, or later on in sperm function. Therefore, it isanticipated that compounds that interfere with the function of thiskinase family could be utilized as contraceptive agents.

Despite the availability of a range of contraceptive methods, over 50%of pregnancies are unintended worldwide and in the United States. Thus,there is a critical need for contraception that better fits the diverseneeds of women and men and takes into consideration different ethnic,cultural and religious values. Except for the use of condoms orvasectomy, the availability of contraceptive methods for men is verylimited.

The importance of protein kinases in most physiological processessuggests that inhibition of tssk kinase family activity with a specificdrug could inhibit fertilization. In accordance with one aspect of thepresent invention, the tssk kinase family is used as a target for thedevelopment of novel drugs. In particular, the sperm-specific tssk geneproducts will be used to screen for specific inhibitors of tssk kinaseactivity and these inhibitors will be used either alone or inconjunction with other contraceptive agents to prevent unintendedpregnancies. Advantageously, the unique sequence of the members of thetssk kinase family supports the likelihood of finding specificinhibitors for their activity. Finally, if these kinases remain in thesperm after spermatogenesis and have a role in sperm physiology, designof specific tssk kinase inhibitors could be used both in male and femalein order to prevent fertilization.

SUMMARY OF THE INVENTION

The present invention is directed to the human sperm-specific tsskkinase gene family and their corresponding proteins. More particularly,the present invention is directed to the human tssk1, tssk2 and tssk 3kinases and the use of those kinases to identify agonists or antagonistsof tssk kinase activity. The present invention also encompassesantibodies raised against the human tssk1, tssk2 and tssk 3 kinases andthe use of these antibodies as diagnostic tools.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison between the amino acid sequences of human tssk 1 (SEQID NO: 4), human tssk 2 (SEQ ID NO: 5), and human tssk 3 (SEQ ID NO: 6).As seen in FIG. 1 the amino acid sequences between the three proteinsdiverge near the carboxy terminus.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, “nucleic acid,” “DNA,” and similar terms also includenucleic acid analogs, i.e. analogs having other than a phosphodiesterbackbone. For example, the so-called “peptide nucleic acids,” which areknown in the art and have peptide bonds instead of phosphodiester bondsin the backbone, are considered within the scope of the presentinvention.

The term “peptide” encompasses a sequence of 3 or more amino acidswherein the amino acids are naturally occurring or synthetic(non-naturally occurring) amino acids. Peptide mimetics include peptideshaving one or more of the following modifications:

-   -   1. peptides wherein one or more of the peptidyl —C(O)NR—        linkages (bonds) have been replaced by a non-peptidyl linkage        such as a —CH₂-carbamate linkage (—CH₂OC(O)NR—), a phosphonate        linkage, a —CH₂-sulfonamide (—CH₂—S(O)₂NR—) linkage, a urea        (—NHC(O)NH—) linkage, a —CH₂-secondary amine linkage, or with an        alkylated peptidyl linkage (—C(O)NR—) wherein R is C₁-C₄ alkyl;    -   2. peptides wherein the N-terminus is derivatized to a —NRR₁        group, to a —NRC(O)R group, to a —NRC(O)OR group, to a —NRS(O)₂R        group, to a —NHC(O)NHR group where R and R₁ are hydrogen or        C₁-C₄ alkyl with the proviso that R and R₁ are not both        hydrogen;    -   3. peptides wherein the C terminus is derivatized to —C(O)R₂        where R₂ is selected from the group consisting of C₁-C₄ alkoxy,        and —NR₃R₄ where R₃ and R₄ are independently selected from the        group consisting of hydrogen and C₁-C₄ alkyl.

Naturally occurring amino acid residues in peptides are abbreviated asrecommended by the IUPAC-IUB Biochemical Nomenclature Commission asfollows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine isIle or I; Methionine is Met or M; Norleucine is Nle; Valine is Vat or V;Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanineis Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine isGln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid isAsp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan isTrp or W; Arginine is Arg or R; Glycine is Gly or G, and X is any aminoacid. Other naturally occurring amino acids include, by way of example,4-hydroxyproline, 5-hydroxylysine, and the like.

Synthetic or non-naturally occurring amino acids refer to amino acidswhich do not naturally occur in vivo but which, nevertheless, can beincorporated into the peptide structures described herein. The resulting“synthetic peptide” contain amino acids other than the 20 naturallyoccurring, genetically encoded amino acids at one, two, or morepositions of the peptides. For instance, naphthylalanine can besubstituted for trytophan to facilitate synthesis. Other synthetic aminoacids that can be substituted into peptides include L-hydroxypropyl,L-3,4-dihydroxyphenylalanyl, alpha-amino acids such asL-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha.-methylalanyl,beta.-amino acids, and isoquinolyl. D amino acids and non-naturallyoccurring synthetic amino acids can also be incorporated into thepeptides. Other derivatives include replacement of the naturallyoccurring side chains of the 20 genetically encoded amino acids (or anyL or D amino acid) with other side chains.

As used herein, the term “conservative amino acid substitution” aredefined herein as exchanges within one of the following five groups:

-   -   I. Small aliphatic, nonpolar or slightly polar residues:        -   Ala, Ser, Thr, Pro, Gly;    -   II. Polar, negatively charged residues and their amides:        -   Asp, Asn, Glu, Gln;    -   III. Polar, positively charged residues:        -   His, Arg, Lys;    -   IV. Large, aliphatic, nonpolar residues:        -   Met Leu, Ile, Val, Cys    -   V. Large, aromatic residues:        -   Phe, Tyr, Trp

As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially freeof contaminants normally associated with the molecule or compound in anative or natural environment.

As used herein, the term “biologically active fragments” or “bioactivefragment” of an tssk polypeptide encompasses natural or syntheticportions of the full-length protein that are capable of specific bindingto their natural ligand.

“Operably linked” refers to a juxtaposition wherein the components areconfigured so as to perform their usual function. For example, controlsequences or promoters operably linked to a coding sequence are capableof effecting the expression of the coding sequence.

The term “non-native promoter” as used herein refers to any promoterthat has been operably linked to a coding sequence wherein the codingsequence and the promoter are not naturally associated (i.e. arecombinant promoter/coding sequence construct).

As used herein, a transgenic cell is any cell that comprises a nucleicacid sequence that has been introduced into the cell in a manner thatallows expression of the a gene encoded by the introduced nucleic acidsequence.

The Invention

The present invention is directed to a family of kinases (the tsskkinase family) that are expressed exclusively in germ cells of humansand mice. The mouse homologs of the human tssk genes were previouslydescribed and were found to be expressed postmeiotically in male germcells. (Bielke et al., 1994, Gene 139, 235-9; Kueng et al., 1997, J CellBiol 139, 1851-9) Using a combination of two yeast hybrid technology andcoimmunoprecipitation, Kueng et al. (1997) found that mouse tssk 1 and 2bind and phosphorylate a protein of 54 Kda, that represents the tssksubstrate. This tssk substrate has been designated tsks. The tsksprotein is also testis-specific and its developmental expressionsuggests that it is postmeiotically expressed in germ cell. The mousecDNA sequence of the tssk substrate was previously reported (Kueng etal., 1997) and was used to search the EST data base. A human ESThomologue AL041339 was found and used to generate sense and antisenseprimers for obtaining the full length clone by 5′ and 3′ RACE usinghuman testis marathon ready cDNA (Clontech, Inc.). The full lengthnucleotide sequence of human tsks is provided as SEQ ID NO: 7 and thededuced protein sequences is provided as SEQ ID NO: 8.

The developmental expression pattern of the tssk kinases, as well as thegeneral relevance of kinases in physiological processes led applicantsto believe that this family of testis-specific kinases has a role inspermatogenesis. The finding that the tssk kinase family and one of theputative substrates are expressed at the same time duringspermatogenesis is relevant to the potential use of these proteins ascontraceptive targets. Accordingly, one aspect of the present inventionis directed to the isolation of the human tssk homologs and their use inisolating contraceptive agents.

Since sperm are transcriptionally and translationally inactive, cloningand characterizing candidate sperm protein kinases at the molecularlevel, requires the use of RNA isolated from male germ cells. RNAtranscripts expressed in the male germ cell lineage might ultimately beimportant in sperm function; however, it is also possible that suchtranscripts function during testicular spermatogenesis. Using thismethodology a unique cDNA was cloned from mouse male germ cells thatencodes a putative protein kinase of the ser/thr protein kinasesubfamily. That kinase (tssk3b) is specifically expressedpostmeiotically in murine male germ cells. In accordance with oneembodiment of the present invention a unique testis-specific mouse tsskgene is provided. The nucleic acid sequence and amino acid sequence oftssk3b is provided as SEQ ID NO 10 and SEQ ID NO: 9, respectively.

The human homologue of tssk3b has also been cloned (and designated humantssk3) and exhibits 98% homology to the putative mouse protein at theprotein level. The nucleotide sequence and amino acid sequence of humantssk3 is provided as SEQ ID NO 3 and SEQ ID NO: 6, respectively.Recently, a similar mouse protein kinase was described and identified asa testis-specific serine kinase 3 (mouse tssk3) (Zuercher et al., 2000,Mech Dev 93, 175-7), a member of a small family of testis-specificprotein kinases (Bielke et al., 1994, Gene 139, 235-9; Kueng et al.,1997, J Cell Biol 139, 1851-9). Despite this homology, both the humantssk3 and the mouse tssk3b cDNAs cloned and described in the presentapplication demonstrate differences with mouse tssk3 in several aminoacids.

Using the predicted mouse tssk 1 and 2 amino acid sequences (asdescribed by Kueng et al., 1997) for the purposes of searching the genebank, a genome sequence of both mouse tssk 1 and tssk2 was obtained. The3′ end and 5′ end of the coding region of these genomic mouse genes werethen used to design sense and antisense primers, respectively, and usedto isolate the human homologs using PCR technology (see Example 2). Theamplified cDNA fragments were cloned using the TOPO TA cloning kit(Invitrogen) and sequenced. An amplified human tssk1 gene wasapproximately 1.3 kb in size and the isolated human tssk 2 gene wasapproximately 1.2 kb in size. The nucleic acid sequence and amino acidsequence of human tssk1 is provided as SEQ ID NO: 1 and SEQ ID NO: 4,respectively. The nucleic acid sequence and amino acid sequence of humantssk2 is provided as SEQ ID NO: 2 and SEQ ID NO: 5, respectively.

In accordance with one embodiment of the present invention a purifiedpolypeptide is provided comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 9, or an amino acid sequencethat differs from SEQ ID NO: 4 or SEQ ID NO: 6 by one or moreconservative amino acid substitutions. More preferably the purifiedpolypeptide comprises an amino acid sequence that differs from SEQ IDNO: 1 or SEQ ID NO: 3 by less than 5 conservative amino acidsubstitutions, and more preferably by 2 or less conservative amino acidsubstitutions.

In one preferred embodiment the purified polypeptide comprises the aminoacid sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. Thesepolypeptides may include additional amino acid sequences to assist inthe purification of recombinantly produced polypeptides. In oneembodiment, the purified polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQID NO: 6 and a peptide tag, wherein the peptide tag is linked to thetssk peptide sequence. Suitable expression vectors for expressing suchfusion proteins and suitable peptide tags are known to those skilled inthe art and commercially available. In one embodiment the tag comprisesa His tag (see Example 4). In another embodiment the purifiedpolypeptide comprises the amino acid sequence of SEQ ID NO: 8 or theamino acid sequence of SEQ ID NO: 8 linked to a peptide tag.

In another embodiment, the present invention is directed to a purifiedpolypeptide that comprises a portion of a tssk polypeptide. Moreparticularly the tssk polypeptide portion consists of natural orsynthetic portions of a full-length polypeptide selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 9that are capable of specific binding to their natural ligand. In oneembodiment the human tssk fragment retains its ability to bind to tsks.

The present invention also encompasses nucleic acid sequences thatencode human tssk. In one embodiment a nucleic acid sequence is providedcomprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 10 or fragments thereof. In another embodiment a purified nucleicacid sequence is provided, selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

The present invention also includes nucleic acids that hybridize understringent or highly stringent conditions (as defined herein) to all or aportion of the nucleotide sequence represented by SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 or SEQ ID NO: 10, or their complements. Thehybridizing portion of the hybridizing nucleic acids is typically atleast 15 (e.g., 20, 25, 30, or 50) nucleotides in length. Hybridizingnucleic acids of the type described herein can be used, for example, asa cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe todetect the expression of the human tssk gene.

Nucleic acid duplex or hybrid stability is expressed as the meltingtemperature or Tm, which is the temperature at which a nucleic acidduplex dissociates into its component single stranded DNAs. This meltingtemperature is used to define the required stringency conditions.Typically a 1% mismatch results in a 1° C. decrease in the Tm, and thetemperature of the final wash in the hybridization reaction is reducedaccordingly (for example, if two sequences having >95% identity, thefinal wash temperature is decreased from the Tm by 5° C.). In practice,the change in Tm can be between 0.5° C. and 1.5° C. per 1% mismatch.

In one embodiment, the present invention is directed to the nucleic acidsequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and nucleic acidsequences that hybridize to those sequences (or their complement) understringent or highly stringent conditions. In accordance with the presentinvention highly stringent conditions are defined as conducting thehybridization and wash conditions at no lower than −5° C. Tm. Stringentconditions are defined as involve hybridizing at 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at 68° C.Moderately stringent conditions include hybridizing at 68° C. in5×SSC/5× Denhardt's solution/1.0% SDS and washing in 3×SSC/0.1% SDS at42° C. Additional guidance regarding such conditions is readilyavailable in the art, for example, by Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; andAusubel et al. (eds.), 1995, Current Protocols in Molecular Biology,(John Wiley & Sons, N.Y.) at Unit 2.10.

The present invention is also directed to recombinant human tssk geneconstructs. In one embodiment, the recombinant gene construct comprisesa non-native promoter operably linked to a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 7 and SEQ ID NO: 10. In one embodiment the recombinantgene construct comprises a non-native promoter operably linked to anucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO: 3. The non-native promoter is preferablya strong constitutive promoter that allows for expression in apredetermined host cell. These recombinant gene constructs can beintroduced into host cells to produce transgenic cell lines thatsynthesize the tssk gene products. Host cells can be selected from awide variety of eukaryotic and prokaryotic organisms, and two preferredhost cells are E. coli and yeast cells.

In accordance with one embodiment, a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQID NO: 10 are inserted into a eukaryotic or prokaryotic expressionvector in a manner that operably links the gene sequences to theappropriate regulatory sequences, and human tssk is expressed in theappropriate eukaryotic or prokaryotic cells host cell. Suitableeukaryotic host cells and vectors are known to those skilled in the art.The baculovirus system is also suitable for producing transgenic cellsand synthesizing the tssk genes of the present invention. One aspect ofthe present invention is directed to transgenic cell lines that containrecombinant genes that express human tssk and fragments of the humantssk coding sequence. As used herein a transgenic cell is any cell thatcomprises an exogenously introduced nucleic acid sequence. Morepreferably the introduced nucleic acid is sufficiently stable in thetransgenic cell (i.e. incorporated into the cell's genome, or present ina high copy plasmid) to be passed on to progeny cells. In one embodimentthe transgenic cell is a human cell and comprises a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 10. More preferably thenucleic acid sequence is selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The present invention alsoincludes non-human transgenic organisms wherein one or more of the cellsof the transgenic organism comprise a recombinant gene that expressesthe human tssk.

The present invention also encompasses a method for producing humantssk. The method comprises the steps of introducing a nucleic acidsequence comprising sequences encoding the human tssk into a host cell,and culturing the host cell under conditions that allow for expressionof the introduced human tssk gene. In one embodiment the promoter is aconditional or inducible promoter, alternatively the promoter may be atissue specific or temporal restricted promoter (i.e. operably linkedgenes are only expressed in a specific tissue or at a specific time).The synthesized tssk can be purified using standard techniques and usedin high throughput sceeens to identify inhibitors of tssk activity.Alternatively, in one embodiment the recombinantly produced tsskpolypeptides, or fragments thereof are used to generate antibodiesagainst the tssk polypeptides. The recombinanatly produced tssks arealso used to obtain crystal structures. Such structures would allow forcrystallography analysis that would lead to the design of specific drugsto inhibit tssk function.

Preferably, the nucleic acid sequences encoding the sperm-specifickinase are inserted into a suitable expression vector in a manner thatoperably links the gene sequences to the appropriate regulatorysequences for expression in the preselected host cell. Suitable hostcells, vectors and methods of introducing the DNA constructs into cellsare known to those skilled in the art. In particular, nucleic acidsequences encoding the sperm-specific kinase may be added to a cell orcells in vitro or in vivo using delivery mechanisms such as liposomes,viral based vectors, or microinjection.

In accordance with one embodiment a composition is provided comprising apeptide having the sequence selected from the group consisting of SEQ IDNO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or an antigenic fragment thereof.In one embodiment the antigenic fragment consists of the sequence of SEQID NO: 4 or SEQ ID NO: 6. The compositions can be combined with apharmaceutically acceptable carrier or adjuvants and administered to amammalian species to induce an immune response.

Another embodiment of the present invention is directed to the isolatedantibodies that are generated against human tssk or fragments thereof.Antibodies to human tssk may be generated using methods that are wellknown in the art. In accordance with one embodiment an antibody isprovided that specifically binds to a polypeptide selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO: 9. In one embodiment antibodies are provided that bindto a polypeptide selected from the group consisting of SEQ ID NO: 4, SEQID NO: 5 and SEQ ID NO: 6. In one preferred embodiment the antibody is amonoclonal antibody. The antibodies may be used with or withoutmodification, and may be labeled by joining them, either covalently ornon-covalently, with a reporter molecule. In addition, the antibodiescan be formulated with standard carriers and optionally labeled toprepare therapeutic or diagnostic compositions.

The present invention also provides a method for detecting the presenceof human tssk. The method comprises the steps of contacting a samplewith a labeled antibody that specifically binds to human tssk, removingunbound and non-specific bond material and detecting the presence of thelabeled antibody. In one embodiment the labeled compound comprises anantibody that is labeled directly or indirectly (i.e. via a labeledsecondary antibody). In particular, the tssk antibodies of the presentinvention can be used to confirm the expression of tssk as well as itscellular location, or in assays to monitor patients being treated withhuman soluble tssk or inhibitors.

In accordance with one aspect of the present invention, the tssk kinasefamily is used as a target for the development of novel drugs. Progressin the field of small molecule library generation using combinatorialchemistry methods coupled to high-throughput screening has acceleratedthe search for ideal cell-permeable inhibitors. In addition,structural-based design using crystallographic methods has improved theability to characterize in detail ligand-protein interaction sites thatcan be exploited for ligand design.

In one embodiment, the present invention provides methods of screeningfor agents, small molecules, or proteins that interact with polypeptidescomprising the sequence of tssk1, tssk2, tssk3 or bioactive fragmentsthereof. As used herein, the term “biologically active fragments” or“bioactive fragment” of tssk1, tssk2, tssk3 encompasses natural orsynthetic portions of the native peptides that are capable of specificbinding to at least one of the natural ligands of the respective nativetssk1, tssk2, tssk3 polypeptide, including tsks. The inventionencompasses both in vivo and in vitro assays to screen small molecules,compounds, recombinant proteins, peptides, nucleic acids, antibodiesetc. which bind to or modulate the activity of tssk1, tssk2, tssk3 andare thus useful as therapeutics or diagnostic markers for fertility.

In one embodiment of the present invention tssk polypeptides, selectedfrom the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6and SEQ ID NO: 9, are used to isolate ligands that bind to tssk underphysiological conditions. The method comprises the steps of contactingthe tssk polypeptides with a mixture of compounds under physiologicalconditions, removing unbound and non-specifically bound material, andisolating the compounds that remain bound to the tssk polypeptides.Typically, the tssk polypeptides will be bound to a solid support, usingstandard techniques, to allow for rapid screening of compounds. Thesolid support can be selected from any surface that has been used toimmobilize biological compounds and includes but is not limited topolystyrene, agarose, silica or nitrocellulose. In one embodiment thesolid surface comprises functionalized silica or agarose beads.Screening for such compounds can be accomplished using libraries ofpharmaceutical agents and standard techniques known to the skilledpractitioner.

Ligands that bind to the tssk polypeptides can then be further analyzedfor agonists and antagonists activity through the use of an in vitrokinase assay as described in Example 7. Inhibitors of tssk kinaseactivity have potential use as agents that preventmaturation/capacitation of sperm. Such inhibitors can be formulated aspharmaceutical compositions and administered to a subject to blockspermatogenesis and provide a means for contraception.

In accordance with one embodiment, specific inhibitors of the human tsskkinase activity are identified through the use of an in vitro kinaseassay that is capable to detecting phosphorylation events. In oneembodiment the method of identifying inhibitors of tssk kinase activitycomprises combining a labeled source of phosphate with one or more ofthe human tssk polypeptides in the presence of one or more potentialinhibitory compounds. As described in Example 4 the tssk proteins havethe property to become autophosphorylated. Therefore by comparing therate of autophosphorylation that occurs in the presence and absence ofthe candidate inhibitory compound, specific inhibitory compounds can beidentified. In one preferred embodiment a tssk substrate is provided andthe assay is based on measuring the rate of phosphorylation of saidsubstrate in the presence and absence of the candidate inhibitorycompounds. Preferably large numbers of compounds will screened usinghigh through put techniques to identify tssk specific inhibitorycompounds.

In accordance with one embodiment specific inhibitors of the tssk kinaseactivity are identified by providing an in vitro kinase assaycomposition, wherein the composition comprises a labeled source ofphosphate, a tssk substrate and a tssk kinase selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 9.The rate of tssk substrate phosphorylation will be determined undercontrolled conditions in the absence of any inhibitory compounds, andthen the identical conditions will be used to measure the rate ofphosphorylation of the tssk substrate when the assay is run in thepresence of one or more potential inhibitory compounds. Those compoundsthat decrease the activity of the tssk kinase will be identified andtested to determine if the inhibitory effect is specific to the tsskkinase.

In one embodiment the method for identifying human tssk inhibitorscomprises the steps of providing a labeled source of phosphate, a tssksubstrate and a tssk kinase selected from the group consisting of SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, contacting that composition with oneor more potential inhibitory compounds and measuring the rate ofphosphorylation. In one preferred embodiment the labeled source ofphosphate is [γ32P] ATP and the tssk substrate comprises an amino acidsequence of SEQ ID NO: 8. The kinase assay can also be conducted withtwo or more of the human tssk kinases present to confirm the activity ofthe inhibitor to all three kinases.

EXAMPLE 1

Isolation of a Novel Mouse Testis Specific Serine/Threonine Kinase

Reverse transcription-polymerase chain reaction (RT-PCR) usingdegenerate oligonucleotides corresponding to conserved regions presentin protein kinases resulted in the isolation of a novel member of thetestis-specific serine/threonine kinase. This PCR fragment recognized a1020 bp transcript in male germ cells by northern blot analysis. Usingthis fragment as a probe, a full length cDNA was cloned from a mousemixed germ cell cDNA library. This cDNA has an open reading frame of 804bases encoding a protein of 268 amino acids. Tissue expression analysisrevealed that this protein kinase is developmentally expressed in mousetesticular germ cells and is not present in brain, ovary, kidney, liveror early embryonic cells.

This novel putative serine/threonine protein kinase (SEQ ID NO: 9) isalmost identical to tssk3 (Zuercher et al., 2000, Mech Dev 93, 175-7), arecently described mouse testis-specific protein kinase, with theexception of several base pair deletions that result in a shift in thecoding region and an alteration of 22 amino acids (residues 109 to 131).The human homologue of this novel protein kinase (SEQ ID NO: 6) wassubsequently cloned and displayed expression exclusively in the testis.Fluorescence in situ hybridization (FISH) using both the human and mousecDNA clones revealed syntenic localization on chromosomes 1p34-35 and4E, respectively. Due to the homology with tssk3 this novel proteinkinase is named tssk3b.

Materials and Methods

Isolation of Spermatogenic Cell Populations

Purified populations of pachytene spermatocytes, round spermatids, andcondensing spermatids were prepared from decapsulated testes of adultmice (CD-1; Charles Rivers) by sequential dissociation with collagenaseand trypsin-DNase 1 (Bellve et al., 1977; Romrell et al., 1976). Thecells were separated into discrete populations by sedimentation velocityat unit gravity in 2-4% BSA gradients in enriched Krebs RingerBicarbonate Medium (EKRB) (Bellve et al., 1977; Romrell et al., 1976).The pachytene spermatocyte and round spermatid populations were each atleast 85% pure, while the condensing spermatid population was ˜40-50%pure (contaminated primarily with anucleated residual bodies and someround spermatids).

RNA Isolation and Northern Blot Analysis

Total RNA from somatic tissues, testis of mice of defined ages, andisolated spermatogenic cells of adult mice was isolated by homogenizingthe cells in 5 M guanidium isothiocyanate, 25 mM sodium citrate, pH 7.2,0.5% Sarkosyl, and 0.1 M 2-mercaptoethanol (Chirgwin et al., 1979).Lysates were centrifuged over cushions of 5.7 M CsCl, 0.1 M EDTA at114,000×g at 20° C. overnight. Pellets were resuspended and extractedwith phenol:chloroform, and the RNA was precipitated with ethanol. Theintegrity of the RNA was verified by ethidium bromide staining of theribosomal RNA on 1% agarose gels. Equivalent amounts of RNA weresubjected to electrophoresis in 1.2% agarose gels containingformaldehyde (Sambrook et al., 1989). RNA was transferred tonitrocellulose paper, baked at 80° C. for 2 hr, and prehybridized in 50%formamide, 5× Denhartdt's, 0.1% SDS, 100 μg/ml Torula RNA, 5×SSPE for aminimum of 1 hr at 42° C. The appropriate DNA probe was generated byPCR, radiolabeled with 32p-dCTP by the random-primed method andincubated with the blots (1×10⁶ cpm/ml) in 50% formamide, 5×Denhartdt's, 0.1% SDS, 100 μg/ml Torula RNA, 5×SSPE with 10% dextransulfate and hybridized at 42° C. overnight. Blots were washed in 2×SSPE,0.1% SDS and then in 0.1×SSPE, 0.1% SDS, both for 2×10 min at roomtemperature. After washing, the filters were air-dried and exposed tofilm at −70° C. with intensifying screens. Northern blots of humantissues were acquired from Clontech and hybridization was performed asdescribed above.

Reverse Transcription-Polymerase Chain Reaction

Reverse transcription-polymerase chain reaction (RT-PCR) was performedusing Superscript II from Gibco-BRL according to the manufacturer'sinstructions, followed by the use of Taq Polymerase from Promega. ThePCR conditions were as follows: 1 cycle of 2 min. at 95° C.; 35 cyclesof 1 min. at 95° C., 1 min at 55° C. and 1 min at 72° C.; the cycleswere finished with 1 cycle of 7 min at 72° C. and then chilled to 4° C.The DNA products were then analyzed on 2% agarose gels. The followingdegenerate primers were used to target conserved regions of tyrosinekinases: for the reverse transcription step, a degenerate antisense20-mer corresponding to the coding subregion IX common to the subfamilyof tyrosine kinases with the addition of a 5′ Eco RI consensus site forsubcloning after three random nucleotides(CGTGGATCCA(A/T)AGGACCA(C/G)AC(A/G)TC; SEQ ID NO: 11); (2) For the PCRstep the same primer was used together with a degenerate sense 20-mercorresponding to the subregion VIb common to the subfamily of tyrosinekinases, also a consensus site for Eco RI was introduced after fourrandom nucleotides ATTCGGATCCAC(A/C)G(A/C/T/G)GA(C/T)(C/T)T; SEQ ID NO:12. After the PCR reaction was completed and the products analyzed, thecDNA was subcloned into a TOPO TA cloning vector according to themanufacturers instructions (In Vitrogen), the Eco RI subcloningconsensus sites were not used. Minipreps from 35 positive colonies wereprepared using a Qiagen kit and then sequenced using a T7 primer.

Cloning of Mouse tssk 3b

To clone tssk 3b, the unique sequence obtained by RT-PCR was used todesign specific primers against this novel kinase. Primers A2(antisense: CATCACCTTTCTTGCTATCATGGG; SEQ ID NO: 13) and S2 (sense:(TGTGAGAACGCCTTGTTGCAG; SEQ ID NO: 14) were used to obtained a PCRfragment of 169 bases. This PCR fragment was subsequently radiolabeledwith 32p-dCTP by the random-primed method and used as a probe to screena oligo-dT primed mixed germ cell cDNA library as previously described.

Cloning of the Human tssk3b Homologue

Using the predicted mouse tssk3b amino acid sequence for the purposes ofsearching the human EST database, entry AI 553938 was pulled out in aBLAST search. This ESTsequence was used to design an antisense primer A3(GACATCACCTTTTTTGCTATCGT; SEQ ID NO: 15). This primer and an adaptorprimer (CCATCCTAATACGACTCACTATAGGGC; SEQ ID NO: 16) were used in 5′ RACEusing human testis marathon ready cDNA as the template (Clontech, Inc).The PCR reaction mix was first heated for 10 min at 94° C. and the mixthen subjected to 40 cycles of PCR under the following condition: 94° C.30 sec, 55° C. 30 sec, 72° C. 2 min. Following these cycles, the mix wasincubated at 72° C. for 10 min. AmpTaq Gold (Perkin Elmer) was used inplace of conventional Taq polymerase for the PCR. The amplified cDNAfragment was cloned and sequenced using the TOPO TA cloning kit(Invitrogen). The 5′ end of the cDNA sequence obtained following 5′ RACEwas used to design a sense primer S3 (GCAGGTGAGAATGTTCTAACGCTG; SEQ IDNO: 17); an antisense primer A4 (TCTCCCCCTACTTTATTGGAGAGC; SEQ ID NO:18) was based on the 3′ end of AI 553938. These two primers were thenused to amplify the full length human tssk3b homologue from the samelibrary using the conditions described above. The amplified 1.058 kbfragment was excised, cloned and sequenced using the University ofVirginia sequencing facility. A C-terminal 400 bp fragment of the cDNAlibrary amplified tssk3b human homologue was used for northern blots ofhuman tissues.

DNA Sequencing and Computer Analysis

All sequencing was performed with the AmpliTaq, FS dye terminator cyclesequencing kit chemistry and the appropriate primers using a 373A DNAsequencer (PE Applied Biosystems, Foster City, Calif.). Ambiguities wereresolved by sequencing the opposite strand. DNA and protein sequenceanalyses were performed using the MacVector™ (Kodak Scientific ImagingSystems, New Haven, Conn.) and Sequencher™ (Gene Codes Corp., Ann Arbor,Mich.) software programs.

Fluorescence In Situ Hybridization and Chromosomal Mapping

Fluorescence in situ hybridization (FISH) and detection ofimmunofluorescence were carried out as previously described (Bell etal., 1995, Cytogenet. Cell Genet, 70: 263-267). The 1 kb, mouse cDNAclone, as well as the corresponding 1 kb human homologue, werebiotinylated by nick translation in a reaction containing 1 μg DNA, 20μM each of dA TP, dCTP and dGTP (Perkin Elmer), 1 μM dTTP (PerkinElmer), 25 mM Tris-HCl, pH 7.5, 5 mM MgC12 (Sigma), 10 mMB-mercaptoethanol (Sigma), 10 μM biotin-16-dUTP (Boehringher Mannheim),2 units DNA polymerase I/DNase I (GIBCO, BRL), and H₂O to a total volumeof 50 μl. Probes were denatured and hybridized to metaphase spreads fromhuman peripheral lymphocytes and mouse embryonic fibroblasts,respectively. The hybridized probe was detected with fluorescein-labeledavidin and the signal amplified by the addition of anti-avidin antibody(Oncor) and a second layer of fluorescein-labeled avidin. The chromosomepreparations were counterstained with DAPI and observed with a ZeissAxiophot epiflourescence microscope equipped with a cooled CCD camera(Photometrics, Tucson, Ariz.) operated by a Macintosh computerworkstation. Digitized images of DAPI staining and FITC signals werecaptured, pseudocolored, and merged using Oncor version 1.6 software.

Results

Cloning of Protein Kinases from Murine Male Germ Cells

Mammalian sperm capacitation is accompanied by an increase in proteintyrosine phosphorylation of several proteins. Since mature sperm are notable to synthesize proteins and the presence of RNA in these cells iscontroversial, in order to identify protein kinases by RT-PCR for thepurposes of ultimately deducing their function, RNA was first isolatedfrom a mouse mixed germ cell population. Degenerate primers targetingconserved regions in the subfamily of tyrosine kinases were employedand, following TA cloning, 27 sequences obtained as described in Methodswere analyzed and compared with sequences in Gene Bank. Although most ofthe sequences match known members of the tyrosine kinase subfamily, someof the sequences obtained matched members of the subfamily of ser/thrprotein kinases.

Between known members of this subfamily such as B-Rafkinase, a sequencewith homology to a novel family of ser/thr protein kinases participatingin spermiogenesis was detected (Kueng et al., 1997, J Cell Biol 139,1851-9). To clone this novel ser/thr kinase, a 169 bases specific PCRfragment corresponding to this sequence was radiolabeled and a mousemixed germ cell cDNA library was screened as described in Methods. Aclone containing a 1.02 kb cDNA insert was obtained (SEQ ID NO: 10) anddesignated tssk-3b following the nomenclature of Kueng et al. Sequenceanalysis revealed a single open reading frame of 804 nucleotidesencoding a 266 amino acids putative ser/thr protein kinase. Thenucleotides flanking the start methionine conform well to Kozakconsensus sequence. The 3′-untranslated region displays apolyadenylation signal 21 nucleotides upstream of the poly(A) tail. Thesequence contains all the expected conserved domains corresponding to aser/thr kinase.

Expression Pattern of Murine tssk3

The expression pattern of murine tssk-3b was investigated by northernblot analysis of total RNA from different mouse tissues using a tssk-3bspecific probe corresponding to the full length transcript of tssk 3b.This probe recognized a single transcript of approximately 1 kbexclusively in the mixed mouse germ cell population prepared asdescribed in Methods. At high exposures it is also possible todistinguish a 1.35 kb transcript also exclusively in germ cells, thistranscript was not observed when the mouse germ cell library wasscreened and could represent a splicing alternative of tssk 3b. Tofurther analyze the expression pattern of tssk-3b in the testis, anorthern blot with RNA obtained from purified germ cells of the adulttestis was probed. tssk-3b mRNA was found to be expressedpostmeiotically in round and condensing spermatids but not in themeiotic pachytene spermatocytes. Mouse testes differentiate at d 11-12of embryonic development and are populated by primordial germ cells. Thefirst spermatogenic wave is initiated a few days after birth andspermatogonia differentiate to early spermatids before puberty. Thedifferentiation to mature sperm is testosterone dependent and occursafter puberty.

To further investigate whether tssk-3b is expressed postmeiotically,total testis RNA was prepared from mice of different postnatal ages (1,3, 7, 10, 15, 20, 24, 30 and adult) and analyzed by northern blotanalysis. Transcription of tssk-3b began between 20 and 24 days afterbirth confirming a postmeiotic expression of this mRNA.

These results demonstrate that the expression pattern of tssk 3b issimilar to that of mouse tssk 1 and tssk 2 (Kueng et al., 1997, J CellBiol 139, 1851-9), suggesting that tssk 3 mRNA expression isdevelopmentally regulated and that its expression is stimulatedpostmeiotically around the onset of spermiogenesis.

To further analyze the expression pattern of tssk 3b, we have performedRT-PCR using specific primers in oocytes, metaphase II-arrested eggs anddifferent stages of preimplantation embryo development. No PCR productwas observed at any of these stages under conditions in which thecorrect sized tssk 3b PCR product could be amplified from mixed germcell total RNA.

Cloning and Expression of the Human Homologue of tssk 3b

Using the mouse tssk 3 sequence, a human EST from germ cell tumor wasidentified by a BLAST search of the databases, and used in conjunctionwith 3′ and 5′ RACE to clone the full length human homologue (SEQ ID NO:3) from an adapted ligated human testis cDNA library (Clontech) asdescribed in Methods.

To investigate the expression pattern of tssk 3 in human tissues, atissue northern blot (Clontech) was probed with a C terminal400 bpfragment of the human tssk3 cDNA. 1 kb and 1.35 kb RNA transcripts wereexpressed exclusively in the testis. Similar to the mouse case, the 1.35kb fragment could represent an alternative spliced transcript. Both themouse tssk 3b and the human homologue tssk 3 of these novel ser/thrkinases have the highest homology (98%) between each other, followed bymouse tssk3 (92%) mouse tssk1 and mouse tssk 2 (56%) suggesting thatthis kinase belongs to the same subfamily of novel ser/thr kinases. Asmentioned, tssk 3 and tssk 3b have a very high homology (92%), thedifference between these two sequences is restricted to a stretch of 22aminoacids (residues 109 to 131). When this stretch is analyzed at thenucleotide level, three base pair deletions were observed in the mousetssk 3 sequence that resulted in a shift in the coding region and analteration of the aforementioned 22 amino acids. It is unclear at thismoment the origin of these frame shifts, one possibility is that twodifferent tssk 3 are present in mouse testis. More likely, one of thesetwo sequences could have a small mistake in the sequence. Since the tssk3 b human homologue was obtained independently from the mouse cDNA cloneand has 100% homology at the amino acid level with its mouse homologue,applicants are confident about the accuracy of both the mouse 3b and thehuman tssk 3 sequences.

Chromosomal Mapping of Human and Murine Tssk3.

The chromosomal location of tssk 3b has also been mapped by fluorescencein situ hybridization (FISH) using the full length human cDNA probe.Fluorescent signals were detected on chromosome 1 in all 20 metaphasespreads scored. Among a total of 109 signals observed, 49 (45%) were on1 p. All chromosome-specific signals were localized to 1 p34.1-34.3. Thedistribution of signals was as follows: 1 chromatid (6 cells), twochromatids (14 cells) and three chromatids (5 cells). The mouse tssk 3bcDNA homologue mapped to the syntenic region in chromosome 4, band E.

EXAMPLE 2

Cloning of the Full Length cDNA of the Human Homologues of the tsskKinase Family and a Partial cDNA Sequence from the tssk Substrate.

Using the predicted mouse tssk 1 and 2 amino acid sequences for thepurposes of searching the gene bank, a genome sequence of both mousetssk 1 and tssk2 was obtained. These genes mapped to chromosome 5 and 22respectively and are intronless. The sequences corresponding to the 3′end and 5′ end of the coding region were used to design sense andantisense primers respectively. The antisense primers were used in 5′RACE and the sense primer in 3′ RACE using human testis marathon readycDNA (Clontech, Inc.) as the template in order to ultimately obtain afull length sequence of both human kinase homologs. The amplified cDNAfragments were cloned using the TOPO TA cloning kit (Invitrogen) andsequenced. To amplify the full length human tssk kinases cDNA, the 5′end of the cDNA sequences obtained following 5′ RACE were used to designa 5′ sense primer. The 3′ end of the cDNA sequences obtained following3′ RACE were used to design antisense primers. These two pairs ofprimers were then used to amplify human tssk1 and 2 from the samelibrary. The amplified sequences 1.3 kb (tssk1) and of 1.2 kb (tssk 2)were subcloned and sequenced. The translated human homologues of thethree members of the tssk kinase family are provided as SEQ ID NOS: 4-6,respectively.

To analyze the specificity of expression, commercial blots depictingseveral human tissues were performed using random primed-labeled probesfrom the full length cDNA of the human tssk 1 and 2. In addition arandom prime-labeled probe from a partial 800 base pair sequence wasused to determine the tissue distribution of the tssk substrate.Northern blots were performed using a commercially available humantissue blot (Clonetech).

Northern blots reveal that tssk 1, 2 and tssk substrate are testisspecific mRNAs. These same blots were stripped to the tssk probes andreprobed with a beta actin sequence, confirming that each of the laneswas equally loaded with RNA. To further analyze the specificity ofexpression, the same probes were used to perform dot blots usingcommercially available mRNA arrays from 76 different human tissues(using the Multiple Tissue Expression (MTE™) Array from Clonetech, Cat#7775-1. The only signal obtained in each of the probed MTEs is in thegrid containing testis RNAs. This experiment confirmed that thesemessages are testis specific in human. In addition, Kueng et al. (1997)demonstrated that tssk 1 and 2 are postmeiotically expressed in mousegerm cells and that these messages are not present in other 11 tissues.

EXAMPLE 3

Immunolocalization and Immunoblotting Experiments

In order to determine if the tssk 1, 2 and 3 kinases and their substrateare present in the testis and/or mature sperm specific antibodiesagainst the recombinant proteins will be generated. Alternatively,anti-peptide antibodies against specific peptides designed from thepredicted amino acid sequence of each cDNA will be made. The specificityof each antibody generated will be tested against the recombinant tssk1, 2 and 3. It is expected that the anti-peptide antibodies designedagainst specific amino acid sequences of each protein will be specific.

Antibodies made against tssk kinases and against the tssk substrate willbe used to analyze for the presence of these kinases in other tissues.For this purpose, Clontech protein Medleys™ of different tissues such asbrain, heart, kidney, liver, lung, ovary, placenta, skeletal muscle andspleen will be tested by western blot using the anti tssk and anti tssksubstrate antibodies. Since mRNAs coding for tssk 1, 2 and 3 are onlypresent in the testis, a similar protein distribution is expected. Sincehuman homologues of tssk kinases have more than 80% homology whencompared with their mouse counterparts, it is expected that antiseraagainst the human recombinant tssk kinases recognize the mousehomologues as well. Thus, the antibodies made against the human tsskkinases will be also tested in mouse tissues.

To generate polyclonal antibodies against the purified recombinantprotein. Rats and rabbits will be used for production of antibodies.Protocol as described by Mandal et al. (1999) will be followed for thispurpose. Antibody titers will be monitored by ELISA and specificity ofthe antibody will be checked by SDS-PAGE and Western blotting analysis.

Sperm Preparation.

In order to perform immunolocalization and immunoblotting experimentshuman sperm will be collected from healthy donors and purified usingPercoll (Pharmacia Biotech, Upsala, Sweden) density gradientcentrifugation as previously described (Naaby-Hansen et al., 1997).Sperm will be then resuspended to a final concentration of 2×10⁷cells/ml.

SDS-PAGE and Immunoblotting.

Sperm and other cell types from different tissues will be pelleted bycentrifugation, washed in 1 ml of phosphate buffered saline (PBS),resuspended in sample buffer (Laemmli, 1970) without mercaptoethanol andboiled for 5 min. After centrifuging, the supernatant will be saved,2-mercaptoethanol will be added to a final concentration of 5%, boiledfor 5 min., and then subjected to 10% SDS-PAGE. Protein concentrationwill be determined by ABC kit from Pierce. Electrophoretic transfer ofproteins to Immobilon P and immunodetection will be carried out aspreviously described (Kalab et al., 1994). Gels will be stained eitherwith silver, coomasie blue or will be transferred to immobilon PVDF(Millipore) and probed with the anti recombinant antibodies.

Immunofluorescence.

To determine the intracellular location of tssk 1, 2 and 3, the specificantibodies against the soluble enzyme will be used in immunofluorescenceexperiments and immunoelectromicroscopy of human testicular tissue andhuman sperm. In addition, if the antibodies recognize the mouseantigens, the localization will be explored by immunofluorescence inmouse germ cells and sperm.

Sperm will be treated in the appropriate experimental conditions, fixedin suspension with a solution of 3% (w/v) paraformaldehyde-0.05% (v/v)glutaraldehyde in PBS for 1 h, washed in PBS at 37 C, and thenpermeabilized with 0.1% (v/v) Triton X-100 in PBS at 37 C for 10 min.The sperm will then be washed in PBS and incubated overnight with serialdilutions (5, 10, 50 and 100) of the appropriate antibody as previouslydescribed (Visconti et al., 1996). After washing the sperm with PBS,they will be incubated with FITC-coupled goat anti mouse IgG and thenattached to poly-lysine-coated microscope slides. Following 3× washeswith PBS, the slides will be mounted with fluoromont and fluorescencewill be assessed. Testicular samples obtained from testicular biopsieswill be processed as previosly described (Westbrook et al., 2000).

EXAMPLE 4

Expression of Recombinant tssk Protein

Since many kinases have been expressed as active molecule in E coli(Bodenbach et al., 1994; Letwin et al., 1992). Advantage will be takenof E coli's relatively simple, easy- to scale-up. Open reading frames ofTSSK1, 2, 3 will be amplified and fused with his tag in pET28b vector(Novagen). The plasmid will be transformed into BL21 DE3 or otherappropriate host strain. Recombinant protein production will be inducedby addition of IPTG to 1 mM final in the cultural medium. Recombinantprotein will be purified from E coli lysate using Ni-NTA column undernative condition. To facilitate crystal formation, highly purifiedprotein is optimal. To purify the protein preparation from above,preparative electrophoresis using PrepCell (BIO-RAD) will be employed.Kinase assay will be conducted before proceeding to crystallography.

To produce recombinant testicular tssk kinases and tssk substrate inbacteria, expression constructs of tssk 1, 2 and 3 as well as for thetssk substrate will be made. The coding region of each of these proteinswill be subcloned in the pET28b expression vector that contains at theC-terminus 6 residues of His-tag. In particular, the constructs will bemade using complete ORF. primers that are designed to create an NdeIsite at the 5′ end and an XhoI site at the 3′ end. The amplifiedproducts will be ligated into the NdeI-XhoI sites of pET28b expressionvector. Since the recombinant protein will be fused with the 6 histidineresidues of the expression vector the expressed protein will be purifiedusing Ni-Histidine bind resin affinity chromatography. Once purified,kinase activity will be evaluated with the tssk substrate to make surethat the enzyme has folded correctly.

TSSK 2 protein was successfully expressed in E. coli and purified. Toexpress tssk 2, the open reading frame was subcloned into a pET28bexpression vector carrying a His-tag. Recombinant protein was producedfollowing induction with IPTG. Bacterially expressed and partiallypurified tssk2 was further purified by preparative gel electrophoresisusing a PrepCell from Biorad. The fractions were collected and analyzedby SDS-PAGE. TSSK substrate was expressed and purified similarly. Thesepurified proteins were used to produce rat polyclonal antibodies usingstandard techniques.

Since multiple kinases have the property to become autophosphorylated invivo and in vitro, purified bacterially expressed recombinant tssk 2 wasassayed for autophosphorylation. The experiment was conducted in thepresence of 40 μM ATP (1 μCi of [³²P] ATP), 10 mM Mg²⁺, phosphatasesinhibitors such as p-nitro-phenyl phosphate and glycerol phosphate andproteases inhibitors (leupeptin and aprotinin 10 μg/ml), the assay wasstopped with sample buffer and tssk 2 was separated in 10% PAGE. Autoradiography of the dried gel showed that recombinant tssk 2 incorporated³²P, suggesting that the bacterially expressed tssk 2 folded correctlyfor phosphorylation to occur and could be used for structural studies

Since not all bacterial expressed recombinant proteins are foldedcorrectly, a similar approach will be taken to express tssk 1, 2 and 3in yeasts. In order to express the tssk kinases and substrate in yeasts,the kinases will be subcloned in pPICZαB vector from Invitrogen(Carlsbad, Calif.) and will be expressed in Pichia pastoris as secretedprotein as well as an intracellular protein. These constructs also havea C-terminal His tag that will allow an easy purification of therecombinant proteins either from the culture media or from the extractedcells.

EXAMPLE 5

Tssk 2 Interacts with Tsks in a Yeast Two-Hybrid System

To demonstrate protein-protein interaction between tssk 2 and tsks a twoyeast hybrid system was utilized. In this experiment, a bait gene (tssk2) was first transformed into the reporter strain as a fusion to theGAL4 DNA binding domain (DNA-BD). A second plasmid that expressed tsksas fusion to the GAL 4 activation domain (AD) was also introduced intothe AH109 reporter strain. Western blots were performed to confirmexpression of fusion proteins in yeast. Interaction between tssk 2 andtsks was observed to promote transcription of the Histidine (HIS) geneand allow for the growth of yeast in His-free medium. Similarly,cotransfection of yeast cells with a first construct expressing a p53fused to the GAL4 DNA binding domain and a second construct expressingthe SV40 Large T antigen fused to the GAL 4 activation domain (AD)demonstrated that p53 and the SV40 Large T antigen interacted andpromoted His gene activation. This interaction was used as a positivecontrol. In contrast, fusion proteins of TSSK2 and p53 with the DNA-BDdid not promote growing when cotransfected with the GAL-AD plasmid.Neither GAL-AD alone nor the fusion proteins between GAL-AD and TSKS orGAL-AD and SV40 Large T antigen had the ability to activatetranscription of the His gene. This experiment demonstrated that tssk 2and the substrate tsks are able to interact, suggesting that the humanhomologues of these proteins behave in a similar way to their mousecounterparts.

To investigate whether tssk 2 and tsks also interact in vivo,capacitated and non capacitated sperm will be extracted with Triton X100 in conditions that protect protein-protein interaction and thenimmunoprecipitation will be conducted with specific antibodies. Theinteraction will be assayed by Western blots with the other antibody ina typical cross immunoprecipitation experiment. Since proteins thatinteract should co-localized, double labeling immunofluorescence will beconducted and co-localization investigated. Although rabbit antibodieshave not yet been obtained for the human tssks, rat anti tssk 2 and ratanti tsks antibodies have been previosly obtained and therefore there isno reason to anticipate any difficulties in obtaining antibodies to thehuman tssks.

EXAMPLE 6

Isolation of the Crystal Structure of Tssk 1, 2, 3 and the tsskSubstrate

Two strategies will be followed for the rationale design oftssk-specific inhibitors. First, an in vitro kinase assay compatiblewith High Throughput screening will be developed. Second, crystalstructure of tssk kinases alone or complexed with their substrate willbe obtained.

For structural studies using X-ray crystallography, it is necessary toobtain approximately 5 mg of the active kinases. This amount of proteinis generally a suitable quantity for initial crystallization trials.Initial trials will be performed on the recombinant intact protein; thisis a common crystallization technique that has proven to be successfulin many cases, including several structural studies of enzymes. Prior tocrystallization screening, the expressed fragment would be checked for asuitable level of enzymatic activity as well as for purity, homogeneityand solubility. Several commercially available crystallization screeningkits, used in conjunction with the hanging drop vapor diffusion method,provide the standard first step in the search for crystal growthconditions. Crystallization screening of the fragment in the presence ofcatalytically required metal ions, substrates and/or inhibitors will becarried out simultaneously with the screen of the apoenzyme.

Crystals will be obtained at 21° C. by equilibrating sitting drops. Forcryodiffraction experiments, the crystals will be transferred to asimilarly buffered solution through three solutions with intermediateconcentrations of these reagents. For diffraction experiments at roomtemperature and lower resolution, Cu Kα radiation from rotating anodeX-ray generators will be used. Final structure will be established afterpreliminary refinement, model improvement and further improvement.

In the case of the tssk kinase family, crystallization in the presenceof their substrate will be attempted in order to establish a molecularbasis of the kinase activity. Once initial crystallization conditionsare obtained, they will be optimized in order to obtain crystals thatdiffract to at least 3.0 Å resolution. Direct phasing of the structurevia the MAD (multiple wavelength anomalous dispersion) and MIR (multipleisomorphous replacement) techniques would be carried out simultaneously,to assure success. Semi-automated model-building and refinementtechniques for phased structures would be utilized to achieve rapidstructural results. Once a structure is obtained, the results will beanalyzed with a view to understanding the unique role of the tssk kinasefamily in spermatogenesis and/or sperm function. An ultimate goal ofthese studies would be the use of the structure as a template for thedesign of specific inhibitors of tssk 1, 2 and/or 3, which may proveuseful as contraceptives.

EXAMPLE 7

Development of a tssk-Specific Kinase Assay.

The design of a specific kinase assay for the tssk family isadvantageous from different points of view. First, an kinase assay willallow for the characterization of the kinetic properties of theseenzymes such as the Km for ATP, divalent cation and substrate. Second,since only active enzymes are suitable for crystallographic studies, akinase assay will confirm the folding of the recombinant proteins.Third, it is desireable to develop an in vitro kinase assay at the labscale to be the basis to a kinase assay appropriate for high throughputscreening of tssk-specific inhibitors.

Development of assays to measure tssk kinase activity will followgeneral characteristics of kinase assays such as presence of substrate,[γ32P]ATP, Mg²⁺ and/or Mn²⁺ and phosphatases inhibitors. First however asource of tssk kinase must be provided. The tssk kinase will begenerated by recombinantly expressing these proteins and purifying theexpressed kinase.

Kinase Assay in Mammalian Cell Extracts.

ORFs of the respective human tssk kinases will be subcloned in a pCMV-HAepitope-tagged mammalian expression vector (Clontech cat #K6003-1).Similarly, the ORF of the tssk substrate will be subcloned in a pCMV-Mycmammalian expression vector (Clontech, same as above). Each of the threeHA-kinases will be coexpressed with the cMyc-tssk substrate in threeseparate COS cell lines for each of the respective human tssks.Coexpression will be validated performing immunofluorescence with therespective antitag antibodies. Antibodies against HA and Myc areavailable from Clontech. Since anti c-Myc is a mouse monoclonal and antiHA is a rabbit polyclonal, it will be possible to analyze whether boththe kinase and the substrate were coexpressed in the same cells.

Proteins will be then extracted with 1% Triton and immunoprecipitationwill be performed using anti HA-tag antibodies. Alternatively, antitssk-kinase/anti-tssk substrate antibodies could be used toimmunoprecipitate the tssk kinases and tssk substrate. Typically theantibodies will be linked to a solid support such as a sepharose bead toassist in the isolation of the target ligand. After precipitation withProtein A sepharose, the pellet will be assayed for kinase activityusing different concentrations of ATP (10 μM, 100 μM and 1 mM), Mg²⁺ orMn²⁺ (100 μM, 1 mM and 10 mM) and 1 μCi of [γ32P] ATP. Thephosphorylated protein will be evaluated following SDS-PAGE separationand autoradiography. The evaluation of kinase activity afterimmunoprecipitation is a frequently used method that allow for specificmeasurement of the activity of one particular kinase (Coso et al., 1995,Cell 81, 1137-46; Moos et al., 1995, Biol Reprod 53, 692-9). Since Kuenget al. (1997) has successfully detected the phosphorylation of tssksubstrate after immunoprecipitation of tssk 1 and 2, it is expected thatit will be possible to measure kinase activity of tssk kinases aftercoexpression in a mammalian system.

In Vitro Kinase Assay.

Each purified enzyme will be mixed with different concentrations of ATP(10 μM, 100 μM and 1 mM), Mg²⁺ or Mn²⁺ (100 μM, 1 mM and 10 mM), 1 μCiof [γ32P] ATP and different concentrations of the purified tssksubstrate (1, 10 and 100 ng/assay). Phosphorylation will be evaluatedfollowing SDS-PAGE separation and autoradiography. It is expected thatif the proteins are correctly folded the phosphorylation of thesubstrate will be easily detected with this methodology.

EXAMPLE 8

Generation of tssk Antibodies

Purified tssk 2 and tsks were used to produce rat polyclonal antibodies.Antisera against recombinant human tssk 1, 2 and 3 as well as to thehuman tssk substrate will be employed to define their tissuedistribution and subcellular localization at the protein level. The ratanti-tssk and anti-tsks antibodies recognized the recombinant proteinand also proteins in sperm and testes with the predicted MW, suggestingthat at least one member of the tssk family and their substrate (tsks)are present in sperm. These antibodies were then used to study theintracellular localization of these proteins in capacitated human sperm.Tssk 2 was observed to localized to the equatorial segment of humansperm. Tsks also localized to the equatorial segment. Neverthelessanti-tsks antibody also recognize proteins present in the anterior headand in the tail. Immunolocalization of these molecules suggests thattssk 2 and tsks are present in similar region of the sperm at least in afraction of the human sperm population. Control experiments wereperformed using rat pre immune serum of the respective antibody. BothWestern blots and immunofluorescence were negative. Although theantibody against tssk 2 was produced against tssk 2, we can not discardthat this antibody recognized other members of the tssk family sincetheir sequences have high homology. However, discrimination between thethree tssk isoenzymes, may be possible by generating antibodies againstthe C-terminal domain that is different between tssk 1 and 2 and is notpresent in tssk 3.

1. A purified polypeptide comprising an amino acid sequence fragment ofSEQ ID NO: 4 having kinase activity wherein said fragment binds to anamino acid sequence of SEQ ID NO:
 8. 2. The polypeptide of claim 1wherein the polypeptide fragment is immobilized on a solid surface.
 3. Acomposition comprising a tssk1 polypeptide that comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 4, a fragmentof SEQ ID NO: 4, and an amino acid sequence that differs from the aminoacid sequence of SEQ ID NO: 4 by one or two conservative amino acidsubstitutions, wherein said tssk1 polypeptide has kinase activity; and atsks polypeptide comprising an amino acid sequence of SEQ ID NO:
 8. 4.The composition of claim 3 wherein the tssk1 polypeptide comprises afragment of SEQ ID NO: 4, wherein the fragment binds to said tskspolypeptide.
 5. The composition of claim 3 wherein the tssk1 polypeptidecomprises the amino acid sequence of SEQ ID NO:
 4. 6. A kinase assay kitcomprising a tssk1 polypeptide that comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, a fragment of SEQ IDNO: 4, and an amino acid sequence that differs from the amino acidsequence of SEQ ID NO: 4 by one or two conservative amino acidsubstitutions, wherein said tssk1 polypeptide has kinase activity; and atsks polypeptide comprising an amino acid sequence of SEQ ID NO:
 8. 7.The kit of claim 6 further comprising a labeled source of phosphate. 8.The kit of claim 7 wherein the labeled source of phosphate is [γ³²P]ATP.
 9. The kit of claim 6 wherein the tssk1 polypeptide comprises theamino acid sequence of SEQ ID NO: 4.